The spectroscopy of biological tissue is complicated by the highly heterogeneous content of the tissue. This heterogeneity precludes easily identifiable spectral features in any of the visible, infrared, nuclear magnetic resonance, or Raman, spectra.
Nonlinear microscopy has shown promise in unraveling the spectral complexity of biological tissue because of the rich collection of material properties accessible using nonlinear spectroscopy, as well as the excellent confinement of the nonlinear effects to the focus volume in a microscope. Nonlinear microscopy underlies such methods as
Coherent anti-Stokes Raman Scattering (CARS) microscopy, described by    Duncan et al., Opt. Lett., 7, p. 350 (1982), and    Cheng et al., J. Opt. Soc. Am. A, 19, p. 1363 (2002);
Nonlinear Interferometric Vibrational Imaging (NIVI), described by    Marks et al., Phys. Rev. Lett., 92, 123905 (2004),    Jones et al., Opt. Lett., 31, p. 1543 (2006),    Marks et al., Appl. Phys. Lett., 85, p. 5787 (2004), and    Bredfeldt et al., Opt. Lett., 30, p. 495 (2005);
two-photon microscopy, described by    Denk et al., Science, 243, p. 73 (1990), and    Helmchen et al., Nature Methods, 2, p. 932 (2005);
second harmonic generation microscopy, described by    Vinegoni et al., Opt. Expr., 12, p. 331 (2004),    Yazdanfar et al., Opt. Expr., 12, p. 2739 (2004), and    Campagnola et al., Biophys. J., 77, p. 3341 (1999).
Each of the above methods has demonstrated the potential of nonlinear microscopy to revolutionize our understanding of biological tissues on the sub-μm scale, and all of the aforesaid references are incorporated herein by reference.
In particular, Raman scattering methods probe the vibrational frequencies of molecular normal modes, and the Raman spectrum of a particular molecule is highly specific to the molecule. Unfortunately, many molecules can share the same vibrational frequencies, so that it may be difficult to distinguish molecules based on measurements at a small number of vibrational frequencies.
Dudovich et al., Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy, Nature, 418, pp. 512-14 (2002) teach that, for materials having more than one vibrational level, “it is possible to improve detection selectivity by tailoring shaped pulses to induce quantum interference [between these excited] levels.” They show (in FIG. 2c) a beat induced between two modes of Raman-excited dichloromomethane. However, no teaching is provided as to how coherence may be provided, systematically or otherwise, among multiple modes of a target species.